Arren Bar-Even explains how the FutureAgriculture project is hoping to boost agricultural productivity and overcome the deficits of natural photorespiration.
The EU-funded FET Open project FutureAgriculture aims to design and engineer novel plant metabolic pathways that overcome the natural inefficiencies and support higher photosynthetic rate and yield.
In today’s world, one in seven people are malnourished. This situation is expected to worsen as the human population continues to increase. The prospect of feeding ten to 15 billion people by the year 2100 is a tremendously challenging task that will only be met by the implementation of drastic measures to increase productivity of agriculture. To add to this, photorespiration represents another big challenge to overcome as it limits plant growth.
The photorespiration challenge
The food chain is based on a fundamental biochemical process known as ‘the carbon fixation’. This process transforms the inorganic carbon found in CO2 into organic matter, feeding all life forms. Most of the carbon in the biosphere (≥95%) is fixed through the Calvin–Benson–Bassham Cycle (CBBC or Calvin cycle), a metabolic pathway formed by a complex network of chemical reactions catalysed by several enzymes. The Calvin cycle can be found in higher plants, algae and many types of bacteria.
Despite being under a strong selective pressure for eons, it still displays inefficiencies related to the enzymes that it employs. For instance, the carboxylating enzyme RuBisCO has the essential role of assimilating carbon dioxide (CO2) into the plant metabolism. As a result, almost every carbon atom in the food that we consume ultimately derives from RuBisCO’s activity. However, RuBisCO is very slow and cannot fully distinguish between CO2 and molecular oxygen. When oxygen mistakenly replaces CO2 as a substrate for RuBisCO’s activity, a toxic waste product, 2-phosphoglycolate (2PG), is produced.
2PG must be recycled back into the Calvin cycle via a costly detoxifying process called photorespiration. Despite this, however, plant photorespiration dissipates energy and releases CO2 back into the atmosphere, thereby counteracting the function of RuBisCO, reducing the effective rate of carbon fixation and lowering agricultural productivity.
The FutureAgriculture approach
FutureAgriculture aims to boost agricultural productivity by designing and engineering plants that directly overcome the deficits of natural photorespiration and support higher photosynthetic rate and yield. The resulting synthetic plants should carry out a more efficient metabolism that bypasses photorespiration without releasing CO2.
The new pathways are designed by combining existing or plausible enzymatic reactions, such as reactions that can potentially be catalysed by well-known enzymes or that follow a well-known mechanism. Computational power and chemical logic should help in the identification of the most promising synthetic pathways – i.e., short pathways with low consumption of cellular resources. Once the necessary enzymes, existing or engineered, have been recruited, the selected routes are first tested in vitro before being engineered in bacteria that support the evolution of the pathway activity, and later in simple photosynthetic organisms, cyanobacteria.
Only the most promising pathways are implemented in higher plants to finally monitor their effects on plant growth and physiology. Plant growth rate and biomass yield are expected to increase under various environmental conditions, thus paving the way for enhanced agricultural productivity of crops like rice, wheat, barley, oat, soybean, cotton, and potato.
The FutureAgriculture consortium integrates diverse skills in computational biology, enzyme directed evolution, biochemistry, microbiology, plant genomics, and plant physiology. The team, composed of four research groups coming from renewed research institutes (Max Planck Institute for Terrestrial Microbiology, Max Planck Institute of Molecular Plant Physiology, Weizmann Institute of Science, and Imperial College London) and two SMEs (IN srl and Evogene), collect the essential skills to cover the whole project. Including the design of the optimised pathways, their test in several platforms and, finally, their implementation in plants.
Over the past three years: from in silico to in vivo
The five-year FutureAgriculture project started in January 2016. During the last three years, the consortium has completed the in silico and in vitro phases of the project and is now moving towards the in vivo phase: the implementation of the best pathways in photosynthetic living organisms, namely cyanobacteria and plants. In the in-silico phase, the consortium has uncovered dozens of possible metabolic pathways to bypass native photorespiration without releasing CO2. Five pathways were selected as highly promising, as they succeeded in recycling the toxic waste product 2PG back into the Calvin cycle with minimal consumption of cellular resources, minimal overlap with natural metabolism, and using enzymes that are either naturally available or are easy to engineer.
The analytical search was possible thanks to a novel software that composes and characterises synthetic pathways, developed by the team of the project co-ordinator, Arren Bar-Even from the Max Planck Institute of Molecular Plant Physiology. For two of the best pathways, the team of Tobias Erb and Dan Tawfik, respectively from the Max Planck Institute for Terrestrial Microbiology and the Weizmann Institute of Science, identified and engineered all of the necessary enzyme to sustain the pathway activity.
Still, the whole consortium is constantly working to further optimise the enzymes necessary for the pathways in order to find the most efficient, fast and precise ensemble of enzymes that are able to work together. The optimisation is carried out first by rational design based on chemical knowledge and then by high-throughput approaches. With the team of enzymes identified, the pathways were then fully reconstructed in vitro, obtaining the first proof of principle that, at least in vitro, they improve CO2-fixation-metabolism and, when transplanted in planta, they have the potential to positively affect plant growth.
Before moving into photosynthetic organisms, the pathways were first introduced into E. coli bacteria using them as a platform for the sequential evolution of the pathways towards higher in vivo activity. This innovative platform was selected among the finalist for the Innovation Radar Prize 2018 under the category ‘Excellence Science’. FutureAgriculture uses cyanobacteria to evaluate the in vivo behavior of the synthetic photorespiration pathways. Their quick life-cycle and simpler compartmentalisation make cyanobacteria an ideal model organism to work with before moving into plants.
Currently, the consortium is refining the details to ensure that the pathways can also be introduced in the selected model organism. The team at the laboratory of Patrik Jones at Imperial College London is working with cyanobacteria to evaluate the in vivo behavior of the synthetic photorespiration pathways and has built a custom CO2 monitor to characterise the advantages of the synthetic pathways over their natural counterparts. The quick life-cycle and simpler compartmentalisation of cyanobacteria make them an ideal model organism to work with before moving into plants. Meanwhile, partners at Evogene are carefully selecting and testing the specification that will allow the correct production of the enzymes in Arabidopsis and Brachypodium model plants. The whole consortium is eager to see the results in vivo as they result from so many years of work and they will help to improve even more the selected pathways.
Future Agriculture’s innovation
FutureAgriculture offers not a simple improvement but a giant leap in agricultural productivity. Its solution can be achieved within a reasonable timeframe and it relies on genetic and metabolic engineering and not on morphological or structural modifications. Furthermore, while the improvement of photosynthetic rate and yield via transgenic approaches has been a hot research topic for many years, these efforts focused on introducing existing pathways into new plant hosts.
FutureAgriculture adopts a radically different approach. Rather than reshuffling and grafting existing enzymes in a fashion that resembles natural evolution and is in line with current metabolic-engineering thinking, the project systematically explores new pathways that cannot be obtained by the mixing-and-matching of existing, natural enzymes.
FutureAgriculture’s approach demands the de novo engineering of new enzymes in order to catalyse metabolic transformations that are still unknown in nature. These synthetic enzymes operate together with existing ones to form new pathways that have been created and optimised by chemical logic. Given the combinatorial nature of metabolic pathways, the addition of one novel reaction dramatically expands the resulting possible pathways. Yet, so far, only a handful of studies have implemented synthetic pathways which harbor novel enzymes. FutureAgriculture takes this strategy to a new level by constructing de novo pathways within the very core of carbon metabolism.